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Obesity-induced overexpression of miR-802 impairs glucose metabolism through silencing of Hnf1b
(2013)
The contribution of altered post-transcriptional gene silencing to the development of insulin resistance and type 2 diabetes mellitus so far remains elusive. Here, we demonstrate that expression of microRNA (miR)-143 and 145 is upregulated in the liver of genetic and dietary mouse models of obesity. Induced transgenic overexpression of miR-143, but not miR-145, impairs insulin-stimulated AKT activation and glucose homeostasis. Conversely, mice deficient for the miR-143–145 cluster are protected from the development of obesity-associated insulin resistance. Quantitative-mass-spectrometry-based analysis of hepatic protein expression in miR-143-overexpressing mice revealed miR-143-dependent downregulation of oxysterol-binding-protein-related protein (ORP) 8. Reduced ORP8 expression in cultured liver cells impairs the ability of insulin to induce AKT activation, revealing an ORP8-dependent mechanism of AKT regulation. Our experiments provide direct evidence that dysregulated post-transcriptional gene silencing contributes to the development of obesity-induced insulin resistance, and characterize the miR-143–ORP8 pathway as a potential target for the treatment of obesity-associated diabetes.
Opioid Analgesia in P450 Gene Cluster Knockout Mice: A Search for Analgesia-Relevant Isoforms
(2015)
Optical Fibers as Dosimeter Detectors for Mixed Proton/Neutron Fields - A Biological Dosimeter
(2023)
In recent years, proton therapy has gained importance as a cancer treatment modality due to its conformality with the tumor and the sparing of healthy tissue. However, in the interaction of the protons with the beam line elements and patient tissues, potentially harmful secondary neutrons are always generated. To ensure that this neutron dose is as low as possible, treatment plans could be created to also account for and minimize the neutron dose. To monitor such a treatment plan, a compact, easy to use, and inexpensive dosimeter must be developed that not only measures the physical dose, but which can also distinguish between proton and neutron contributions. To that end, plastic optical fibers with scintillation materials (Gd₂O₂S:Tb, Gd₂O₂S:Eu, and YVO₄:Eu) were irradiated with protons and neutrons. It was confirmed that sensors with different scintillation materials have different sensitivities to protons and neutrons. A combination of these three scintillators can be used to build a detector array to create a biological dosimeter.
Thin films of poly(ethyleneterephthalate) [PET]were exposed to radiation dose ranging from 10 to 30 kGy by using gamma rays in the range 12.8-177.8 MGy using swift light ions of hydrogen. There was no effect of the radiation dose on the optical behaviour of PET as a result of exposure to radiation dose up to 30 kGy brought about by gamma rays but a significant decrease in the optical band gap values was observed when PET was exposed to swift light ions of hydrogen. The data obtained are discussed in terms of optical studies carried out on PET using swift heavy ions.
The immobilization of NAD+-dependent dehydrogenases, in combination with a diaphorase, enables the facile development of multiparametric sensing devices. In this work, an amperometric biosensor array for simultaneous determination of ethanol, formate, d- and l-lactate is presented. Enzyme immobilization on platinum thin-film electrodes was realized by chemical cross-linking with glutaraldehyde. The optimization of the sensor performance was investigated with regard to enzyme loading, glutaraldehyde concentration, pH, cofactor concentration and temperature. Under optimal working conditions (potassium phosphate buffer with pH 7.5, 2.5 mmol L-1 NAD+, 2.0 mmol L-1 ferricyanide, 25 °C and 0.4% glutaraldehyde) the linear working range and sensitivity of the four sensor elements was improved. Simultaneous and cross-talk free measurements of four different metabolic parameters were performed successfully. The reliable analytical performance of the biosensor array was demonstrated by application in a clarified sample of inoculum sludge. Thereby, a promising approach for on-site monitoring of fermentation processes is provided.
High gradient magnetic separation (HGMS) has been established since the early 1970s. A more recent application of these systems is the use in bioprocesses. To integrate the HGMS in a fermentation process, it is necessary to optimize the separation matrix with regard to the magnetic separation characteristics and permeability of the non-magnetizable components of the fermentation broth. As part of the work presented here, a combined fluidic and magnetic force finite element model simulation was created using the software COMSOL Multiphysics and compared with separation experiments. Finally, as optimal lattice orientation of the separation matrix, a transversal rhombohedral arrangement was defined. The high suitability of the new filter matrix has been verified by separation experiments.
Bacillus subtilis and Bacillus licheniformis are widely used for the large-scale industrial production of proteins. These strains can efficiently secrete proteins into the culture medium using the general secretion (Sec) pathway. A characteristic feature of all secreted proteins is their N-terminal signal peptides, which are recognized by the secretion machinery. Here, we have studied the production of an industrially important secreted protease, namely, subtilisin BPN′ from Bacillus amyloliquefaciens. One hundred seventy-three signal peptides originating from B. subtilis and 220 signal peptides from the B. licheniformis type strain were fused to this secretion target and expressed in B. subtilis, and the resulting library was analyzed by high-throughput screening for extracellular proteolytic activity. We have identified a number of signal peptides originating from both organisms which produced significantly increased yield of the secreted protease. Interestingly, we observed that levels of extracellular protease were improved not only in B. subtilis, which was used as the screening host, but also in two different B. licheniformis strains. To date, it is impossible to predict which signal peptide will result in better secretion and thus an improved yield of a given extracellular target protein. Our data show that screening a library consisting of homologous and heterologous signal peptides fused to a target protein can identify more-effective signal peptides, resulting in improved protein export not only in the original screening host but also in different production strains.
In proton therapy, the dose from secondary neutrons to the patient can contribute to side effects and the creation of secondary cancer. A simple and fast detection system to distinguish between dose from protons and neutrons both in pretreatment verification as well as potentially in vivo monitoring is needed to minimize dose from secondary neutrons. Two 3 mm long, 1 mm diameter organic scintillators were tested for candidacy to be used in a proton–neutron discrimination detector. The SCSF-3HF (1500) scintillating fibre (Kuraray Co. Chiyoda-ku, Tokyo, Japan) and EJ-260 plastic scintillator (Eljen Technology, Sweetwater, TX, USA) were irradiated at the TRIUMF Neutron Facility and the Proton Therapy Research Centre. In the proton beam, we compared the raw Bragg peak and spread-out Bragg peak response to the industry standard Markus chamber detector. Both scintillator sensors exhibited quenching at high LET in the Bragg peak, presenting a peak-to-entrance ratio of 2.59 for the EJ-260 and 2.63 for the SCSF-3HF fibre, compared to 3.70 for the Markus chamber. The SCSF-3HF sensor demonstrated 1.3 times the sensitivity to protons and 3 times the sensitivity to neutrons as compared to the EJ-260 sensor. Combined with our equations relating neutron and proton contributions to dose during proton irradiations, and the application of Birks’ quenching correction, these fibres provide valid candidates for inexpensive and replicable proton-neutron discrimination detectors
Paracoccus denitrificans for the effluent recycling during continuous denitrification of liquid food
(2010)
Nitrate is an undesirable component of several foods. A typical case of contamination with high nitrate contents is whey concentrate, containing nitrate in concentrations up to 25 l. The microbiological removal of nitrate by Paracoccus denitrificans under formation of harmless nitrogen in combination with a cell retention reactor is described here. Focus lies on the resource-conserving design of a microbal denitrification process. Two methods are compared. The application of polyvinyl alcohol-immobilized cells, which can be applied several times in whey feed, is compared with the implementation of a two step denitrification system. First, the whey concentrate's nitrate is removed by ion exchange and subsequently the eluent regenerated by microorganisms under their retention by crossflow filtration. Nitrite and nitrate concentrations were determined by reflectometric color measurement with a commercially available Reflectoquant® device. Correction factors for these media had to be determined. During the pilot development, bioreactors from 4 to 250 mg·L-1 and crossflow units with membrane areas from 0.02 to 0.80 m2 were examined. Based on the results of the pilot plants, a scaling for the exemplary process of denitrifying 1,000 tons per day is discussed.
The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) are closely related nuclear receptors involved in drug metabolism and play important roles in the mechanism of phenobarbital (PB)-induced rodent nongenotoxic hepatocarcinogenesis. Here, we have used a humanized CAR/PXR mouse model to examine potential species differences in receptor-dependent mechanisms underlying liver tissue molecular responses to PB. Early and late transcriptomic responses to sustained PB exposure were investigated in liver tissue from double knock-out CAR and PXR (CARᴷᴼ-PXRᴷᴼ), double humanized CAR and PXR (CARʰ-PXRʰ), and wild-type C57BL/6 mice. Wild-type and CARʰ-PXRʰ mouse livers exhibited temporally and quantitatively similar transcriptional responses during 91 days of PB exposure including the sustained induction of the xenobiotic response gene Cyp2b10, the Wnt signaling inhibitor Wisp1, and noncoding RNA biomarkers from the Dlk1-Dio3 locus. Transient induction of DNA replication (Hells, Mcm6, and Esco2) and mitotic genes (Ccnb2, Cdc20, and Cdk1) and the proliferation-related nuclear antigen Mki67 were observed with peak expression occurring between 1 and 7 days PB exposure. All these transcriptional responses were absent in CARᴷᴼ-PXRᴷᴼ mouse livers and largely reversible in wild-type and CARʰ-PXRʰ mouse livers following 91 days of PB exposure and a subsequent 4-week recovery period. Furthermore, PB-mediated upregulation of the noncoding RNA Meg3, which has recently been associated with cellular pluripotency, exhibited a similar dose response and perivenous hepatocyte-specific localization in both wild-type and CARʰ-PXRʰ mice. Thus, mouse livers coexpressing human CAR and PXR support both the xenobiotic metabolizing and the proliferative transcriptional responses following exposure to PB.
The subtilase family (S8), a member of the clan SB of serine proteases are ubiquitous in all kingdoms of life and fulfil different physiological functions. Subtilases are divided in several groups and especially subtilisins are of interest as they are used in various industrial sectors. Therefore, we searched for new subtilisin sequences of the family Bacillaceae using a data mining approach. The obtained 1,400 sequences were phylogenetically classified in the context of the subtilase family. This required an updated comprehensive overview of the different groups within this family. To fill this gap, we conducted a phylogenetic survey of the S8 family with characterised holotypes derived from the MEROPS database. The analysis revealed the presence of eight previously uncharacterised groups and 13 subgroups within the S8 family. The sequences that emerged from the data mining with the set filter parameters were mainly assigned to the subtilisin subgroups of true subtilisins, high-alkaline subtilisins, and phylogenetically intermediate subtilisins and represent an excellent source for new subtilisin candidates.
This book is based on a multimedia course for biological and chemical engineers, which is designed to trigger students' curiosity and initiative. A solid basic knowledge of thermodynamics and kinetics is necessary for understanding many technical, chemical, and biological processes.
The one-semester basic lecture course was divided into 12 workshops (chapters). Each chapter covers a practically relevant area of physical chemistry and contains the following didactic elements that make this book particularly exciting and understandable:
- Links to Videos at the start of each chapter as preparation for the workshop
- Key terms (in bold) for further research of your own
- Comprehension questions and calculation exercises with solutions as learning checks
- Key illustrations as simple, easy-to-replicate blackboard pictures
Humorous cartoons for each workshop (by Faelis) additionally lighten up the text and facilitate the learning process as a mnemonic. To round out the book, the appendix includes a summary of the most popular experiments in basic physical chemistry courses, as well as suggestions for designing workshops with exhibits, experiments, and "questions of the day."
Suitable for students minoring in chemistry; chemistry majors are sure to find this slimmed-down, didactically valuable book helpful as well. The book is excellent for self-study.
Preclinical development of highly effective and safe DNA vaccines directed against HPV 16 E6 and E7
(2011)
Persistent infection with high-risk human papillomaviruses (hrHPV) can result in the formation of anogenital cancers. As hrHPV proteins E6 and E7 are required for cancer initiation and maintenance, they are ideal targets for immunotherapeutic interventions. Previously, we have described the development of DNA vaccines for the induction of HPV16 E6 and E7 specific T cell immunity. These vaccines consist of ‘gene-shuffled’ (SH) versions of HPV16 E6 and E7 that were fused to Tetanus Toxin Fragment C domain 1 (TTFC) and were named TTFC-E6SH and TTFC-E7SH. Gene-shuffling was performed to avoid the risk of inducing malignant transformation at the vaccination site. Here, we describe the preclinical safety evaluation of these candidate vaccines by analysis of their transforming capacity in vitro using established murine fibroblasts (NIH 3T3 cells) and primary human foreskin keratinocytes (HFKs). We demonstrate that neither ectopic expression of TTFC-E6SH and TTFC-E7SH alone or in combination enabled NIH 3T3 cells to form colonies in soft agar. In contrast, expression of HPV16 E6WT and E7WT alone or in combination resulted in effective transformation. Similarly, retroviral transduction of HFKs from three independent donors with both TTFC-E6SH and TTFC-E7SH alone or in combination did not show any signs of immortalization. In contrast, the combined expression of E6WT and E7WT induced immortalization in HFKs from all donors. Based on these results we consider it justified to proceed to clinical evaluation of DNA vaccines encoding TTFC-E6SH and TTFC-E7SH in patients with HPV16 associated (pre)malignancies.